The ovarian cancer cell line SKOV3 was cultivated in McCoy’s 5A Medium (GE Healthcare, PAA Laboratories, Pasching, Austria) with 10% foetal bovine serum (PAA Laboratories) till 90% confluency. After washing in PBS, the cells were scratched with a cell scraper and homogenised in extraction buffer (50 mM HEPES (ph 7.5), 1 mM EGTA, 50 mM KCl, 2 mM MgCl2, 5 mM Mercaptoethanol and 1 μl protease inhibitor cocktail without Leupeptin (P8849, Sigma-Aldrich) per 100 μl extraction-buffer). The solution was stored 30 min on ice before centrifugation at 1000 g for 10 min at 4 °C. Equal amounts of protein lysates were incubated with different treatments for 60 min at 30 °C: without adjunct, with 1 mM CaCl2, with 1 mM CaCl2 and 0.3 U (Units) μ-Calpain (C6108, Sigma-Aldrich), and with 1 mM CaCl2, 0.3 U μ-Calpain and 10 mM Calpeptin (CAS 117591-20-5, Calbiochem, Darmstadt, Germany). Treatment was stopped by addition of 2 × sample buffer (62.5 mm Tris-HCL (pH 6.8), 4% SDS, 10% glycerol) and 10-min incubation at 100 °C. In addition to SKOV3, three exemplary lysates of tumour tissue from the intraperitoneal group (T5213, T6362, T6570) were treated in the same way. After treatment, the protein lysates were investigated in western blotting analysis with the same monoclonal E-Cad antibody (24E10).
Mccoy s 5a medium
Manufactured by GE Healthcare
Sourced in Austria
McCoy's 5A medium is a cell culture media formulation used for the growth and maintenance of various cell types. It provides the necessary nutrients and growth factors for cells to thrive in an in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Foetal bovine serum (fbs), by GE Healthcare (1 mentions)
Super electroporator nepa21 type 2, by Nepa Gene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Rpmi 1640 medium, by GE Healthcare (1 mentions)
Os rc 2, by GE Healthcare (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
F2442, by Merck Group (1 mentions)
Dab horseradish peroxidase color development kit p0203, by Beyotime (1 mentions)
Luteolin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Hematoxylin staining solution, by Beyotime (1 mentions)
Beyoecl plus p0018, by Beyotime (1 mentions)
D5796 500ml, by Merck Group (1 mentions)
9 protocols using mccoy s 5a medium
1
Ovarian Cancer Cell Line Proteolysis
2
Synchronized Cell Cycle Study with Plk4 Inhibition
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HCT116 cells were cultured in McCoy’s 5A medium (GE Healthcare) supplied with 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. U2OS cells were cultured in DMEM supplied with 10% FBS and 1% penicillin/streptomycin. For synchronization of the cell cycle, 2 mM thymidine was added to the medium, as indicated in Fig. S1 A. For Plk4 inhibition, cells were incubated for 4 h in the presence of 200 nM centrinone before fixation.
Cells were transfected with DNA or siRNA using Lipofectamine 2000 or Lipofectamine RNAiMAX, according to the manufacturer’s instructions.
The HCT116 Plk4–mClover cell line was produced via CRISPR-Cas9 genome editing. The mClover sequence was inserted into the 3′ region of the Plk4 gene, and cell clones were selected using hygromycin. Cloned cells were genotyped using PCR, and the proper localization of expressed Plk4–mClover was verified via immunofluorescence. We were only able to obtain a monoallelic cell line, which we therefore used in this study.
Cells were transfected with DNA or siRNA using Lipofectamine 2000 or Lipofectamine RNAiMAX, according to the manufacturer’s instructions.
The HCT116 Plk4–mClover cell line was produced via CRISPR-Cas9 genome editing. The mClover sequence was inserted into the 3′ region of the Plk4 gene, and cell clones were selected using hygromycin. Cloned cells were genotyped using PCR, and the proper localization of expressed Plk4–mClover was verified via immunofluorescence. We were only able to obtain a monoallelic cell line, which we therefore used in this study.
3
Electroporation and Microtubule Destabilization
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COS-7 (CRL-1651; ATCC) cells were grown in phenol red–free DMEM (Gibco) + 10% FBS (Gibco) in a standard mammalian cell incubator. U2OS (HTB-96; ATCC) cells were grown in phenol red–free McCoy’s 5A medium (GE Healthcare) + 10% FBS. U2OS cells stably expressing GFP-Sec61β were provided by T. Rapoport (Harvard University, Boston, MA). All cells were cultured using 0.05% trypsin (Gibco) to split and subculture. DNA plasmids were transfected using Super Electroporator NEPA21 Type II (Nepa Gene). Electroporation was performed on 106 cells that were suspended in OptiMEM (Gibco) and 1–10 µg DNA, depending on the desired expression level, in electroporation cuvettes that had a 2-mm gap (12358-346; Bulldog Bio). Cells were electroporated using the following program: 125-V poring pulse, 3-ms pulse length, 50-ms pulse interval, two pulses, with decay rate of 10% and + polarity, followed by a 25-V transfer pulse, 50-ms pulse length, 50-ms pulse interval, five pulses, with a decay rate of 40% and ± polarity. Cells were imaged 12–48 h after electroporation.
In microtubule destabilization experiments, cells were treated with 33 µM nocodazole for 30 min at 37°C in a mammalian cell incubator. Samples were imaged for up to 30 min in the presence of nocodazole so no cells were exposed to 33 µM nocodazole for >1 h.
In microtubule destabilization experiments, cells were treated with 33 µM nocodazole for 30 min at 37°C in a mammalian cell incubator. Samples were imaged for up to 30 min in the presence of nocodazole so no cells were exposed to 33 µM nocodazole for >1 h.
4
Cultivation of Human RCC Cell Lines
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Human RCC cell lines (Caki-1, 786-O, ACHN, 769-P and OS-RC-2) were obtained from the Cell Type Culture Collection in the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Caki-1 cells were cultured in McCoy's 5A medium (HyClone; GE Healthcare Life Sciences), while 786-O, 769-P and OS-RC-2 cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences), and ACHN cells was cultured in minimum essential media (HyClone; GE Healthcare Life Sciences) at 37°C with 5% CO2. All the culture media were supplemented with 10% foetal bovine serum (HyClone; GE Healthcare Life Sciences). 786-O and Caki-1 cell lines were authenticated, and no mycoplasma contamination was identified.
5
Colon Cancer Cell Lines and Normal Tissue Study
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Colon cancer cell lines (HT29, RKO) and the normal mammary epithelial cell line HEK293 were used. We cultured cell lines with McCoy's 5A medium (GE Healthcare, Chicago, IL, USA) or Dulbecco's modified Eagle's medium (Gibco Laboratories, Rockville, MD, USA) containing 10% fetal bovine serum. Human CRC samples and adjacent normal tissues were acquired from the Sir Run Run Shaw Hospital (Hangzhou, Zhejiang, China) between February 2005 and June 2006. All procedures were supervised and approved by the ethics committee of Sir Run Run Shaw Hospital.
6
Subcellular Protein Extraction and SiMPull Assay
7
Colon Cancer Cell Lines and Colorectal Cancer Samples
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A group of colon cancer cell lines (COLO320, SW620, SW480, LOVO, HCT116, DLD1, SW48, HT29, RKO) was used. The cells were cultured in McCoy's 5A Medium (GE, Healthcare, PAA Laboratories, Pasching, Austria) or Dulbecco's Modified Eagle's Medium (DMEM) (Gibco BRL, Rockville, MD, U.S.) with 10% fetal bovine serum.
125 primary colorectal cancer patients who underwent surgery between February 2004 and June 2006 at the Sir Run Run Shaw Hospital (Hangzhou, Zhejiang, China) were investigated for the immunohistochemistry experiments. Patients in this study had not received preoperative chemotherapy. In addition, we used 10 normal colon mucosa biopsy samples as normal controls and 29 CRC cases for MSP and Western blot. This study was approved by the ethics committee of Sir Run Run Shaw Hospital, Zhejiang University.
125 primary colorectal cancer patients who underwent surgery between February 2004 and June 2006 at the Sir Run Run Shaw Hospital (Hangzhou, Zhejiang, China) were investigated for the immunohistochemistry experiments. Patients in this study had not received preoperative chemotherapy. In addition, we used 10 normal colon mucosa biopsy samples as normal controls and 29 CRC cases for MSP and Western blot. This study was approved by the ethics committee of Sir Run Run Shaw Hospital, Zhejiang University.
8
Quantitative Analysis of MMPs
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DMEM and McCoy's 5A medium were purchased from GE Healthcare Life Sciences, HyClone Laboratories. Histostain-Plus Kits (SP-9001, SP-9002) and Mouse Anti-β actin mAb(TA-09) were purchased from ZSGB-Bio, Beijing, China. Luteolin (≥98%, L9283) and ellagic acid (≥95%, E2250) were purchased from Sigma-Aldrich (USA). DAB Horseradish Peroxidase Color Development Kit (P0203), BeyoECL Plus (P0018) and Hematoxylin Staining Solution (C0107) were purchased from Beyotime Institute of Biotechnology. MMP2 (BMO569) and MMP9 (PB0709) were purchased as primary antibodies from Boster Biological Technology Co., LTD.46 (link) HRP-linked rabbit- and mouse-anti IgG (7074s, 7076s) were chosen from CST (USA). Mouse MMPs ELISA Kit (DM-X6142, DM-X6008) was purchased from Baomanbio, Shanghai, China.
9
Genetic Engineering and Cell Line Maintenance
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HeLa and U2OS cells were maintained in DMEM (D5796-500ML; Sigma-Aldrich) supplemented with 10% FBS. HCT116 cells were maintained in McCoy's 5A medium (SH30200.01; GE Healthcare) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Genetic engineering in HCT116 cells was undertaken as described previously (Natsume et al., 2016 (link)). To induce the degradation of RIF1-mAID, 0.1 µg/ml doxycycline and 100 µM indole-3-acetic acid (IAA, a natural auxin; Nacalai Tesque) were added to the culture medium. For the degradation of MCM2-mAID, 500 µM IAA was added to the culture medium. The DR-GFP (Maria Jasin, Memorial Sloan Kettering Cancer Center, New York, NY) or pEJ reporter gene were stably integrated into the genomes of HeLa and U2OS cells. siRNA sequences were as follows: siNBS1 #1, 5′-GUACGUUGUUGGAAGGAAA-3′; siNBS1 #2, 5′-GGGAAAGGGAUGAAGAAAA-3′; siNBS1 #3, 5′-GGACACAAAACCAGAGUUA-3′; siRIF1, 5′-GAAUGAGCCCCUAGGGAAA-3′; siMCM2, 5′-UCAUCGGAAUCCUUCACCA-3′; siBRCA1, siGENOME SMART pool M-003461-02 (Thermo Fisher Scientific).
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